Pivotal role of calnexin and mannose trimming in regulating the endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain

We have established a mammalian semipermeabilized cell system that faithful ly reconstitutes the proteasome-mediated degradation of major histocompatib ility complex Class I heavy chain. We show that degradation required unfold ing of the protein and was cytosol- and ATP-dependent and that dislocation and degradation required proteasome activity. When the interaction of heavy chain with calnexin was prevented, the rate of degradation was accelerated , suggesting that an interaction with calnexin stabilized heavy chain. Stab ilization of heavy chain to degradation was also achieved either by prevent ing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensur e degradation of heavy chain. When degradation or mannose trimming was inhi bited, heavy chain formed a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken toget her, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of g lycoproteins entering the secretory pathway.